This application claims priority under 35 U.S.C. xc2xa7119 to U.K. Patent Application No. 9820525.5, filed Sep. 21, 1998.
The present invention relates to a novel formulation particularly, but not exclusively, for use in immunization.
The immune system has evolved specifically to detect and eliminate foreign or new material from a host. This material may be of viral, bacterial, or parasitic origin and may reside outside or within the cells of the host, or be of neoplastic origin.
The immune response to antigen is generally either cell mediated (T-cell mediated killing) or humoral (antibody production via recognition of whole antigen). The pattern of cytokine production by TH (T-Helper) cells involved in an immune response can influence which of these response types predominates: cell mediated immunity (TH1) is characterized by high IL-2 and IFNxcex3 but low IL-4 production, whereas in humoral immunity (TH2) the pattern is low IL-2 and IFNxcex3 but high IL-4, IL-5, IL-10. Since the secretory pattern is modulated at the level of the secondary lymphoid organ or cells, pharmacological manipulation of the specific TH cytokine pattern can influence the type and extent of the immune response generated.
The TH1xe2x88x92TH2 balance refers to the interconversion of the two different forms of helper T cells. The two forms have large scale and opposing effects on the immune system. If an immune response favors TH1 cells, then these cells will drive a cellular response, whereas TH2 cells will drive an antibody-dominated response. The type of antibodies responsible for some allergic reactions is induced by TH2 cells.
Vaccination is the best known and most successful application of immunological principles to human health. Naturally, to be introduced and approved, a vaccine must be effective and the efficacy of all vaccines is reviewed from time to time. Many factors affect vaccine efficacy. An effective vaccine must: induce the right sort of immunity; be stable on storage; and have sufficient immunogenicity. With non-living vaccines, in particular, it is often necessary to boost their immunogenicity with an adjuvants. This can also apply to some live, e.g., attenuated, vaccines. An xe2x80x9cadjuvantsxe2x80x9d is a substance that enhances the immune response to an antigen.
During work in the 1920s on the production of animal antisera for human therapy, it was discovered that certain substances, notably aluminum salts, added to or emulsified with an antigen, greatly enhance antibody production, i.e., they act as adjuvants. Aluminum hydroxide is still widely used with, for example, diphtheria and tetanus toxoids.
GB-A-1 377 074, corresponding to U.S. Pat. No. 3,792,159, describes a process for preparing coprecipitates of tyrosine having an allergen dispersed therein.
GB-A-1 492 973, corresponding to U.S. Pat. No. 4,070,455, describes a process for preparing coprecipitates of tyrosine having a modified allergen dispersed therein. The allergen is modified by treatment with an agent, such as glutaraldehyde, which causes intra-molecular cross-linking and reduces the allergenicity of the product relative to the unmodified allergen.
3 De-O-acylated monophosphoryl lipid A (3-DMPL) is known from GB-A-2 220 211, corresponding to U.S. Pat. No. 4,912,094 and assigned to Ribi Immunochem. Res. (xe2x80x9cRibixe2x80x9d). Chemically, 3-DMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem. Montana. A preferred form of 3 de-O-acylated monophosphoryl lipid A is disclosed in International Patent Publication No. WO 92/16556.
International Patent Publication No. WO 98/44947 describes a formulation for use in desensitization therapy of allergy sufferers that comprises an optionally modified allergen, tyrosine and 3 de-O-acylated monophosphoryl lipid A.
Considerable efforts have been made to produce better adjuvants, particularly for T-cell-mediated responses, but it should be stressed that very few of these more recent adjuvants have been accepted for routine human use.
It appears that the effect of adjuvants is due mainly to two activities: the concentration of antigen in a site where lymphocytes are exposed to it (the xe2x80x9cdepotxe2x80x9d effect) and the induction of cytokines, which regulate lymphocyte function. Newer antigen delivery systems such as liposomes and immune-stimulating complexes (ISCOMS) achieve the same purpose by ensuring that antigens trapped in them are delivered to antigen-presenting cells. Bacterial products such as mycobacterial cell walls, endotoxins, etc., are believed to act by stimulating the formation of cytokines. Cytokine induction may be particularly useful in immunocompromised patients, who often fail to respond to normal vaccines. It is hoped that such cytokine induction might also be useful in directing the immune response in the desired direction, e.g., in diseases where only TH1 or TH2 cell responsiveness is wanted (Roitt et al. xe2x80x9cImmunology,xe2x80x9d 4th edition Wolfe Publishing, 1995).
We now provide a new antigen formulation that can tilt the TH1-TH2 balance in favor of a TH1 response. The formulation is useful in immunotherapy, particularly the field of vaccines. It is also useful in studying immune responses and in the production of antibodies.
According to one aspect of the present invention there is provided a composition comprising:
(a) an antigen;
(b) a TH1-inducing adjuvants; and
(c) a sparingly soluble amino acid or a derivative thereof.
Preferably, the antigen is derived from a bacterium or virus, or other pathogenic organism, or neoplasm or from knowledge of their antigenic structures.
Preferably, the TH1-inducing adjuvants is monophosphoryl lipid A (MPL), 3xe2x80x2-de-O-acetylated monophosphoryl lipid A (3-DMPL), or a derivative or salt thereof
Preferably, the sparingly soluble amino acid is tyrosine, tryptophan or a derivative thereof.
The present invention also provides a composition for use in medicine. That is, the invention provides a method for using a composition to treat or prevent or reduce susceptibility to a bacterial infection, a viral infection or other disease such as cancer.
Preferably, the composition is in the form of a vaccine, and the invention further provides a method for preparing an immunoglobulin, comprising immunizing an animal with a composition of the present invention.
The present invention also provides a method for preparing a composition of the present invention comprising mixing a solution of an antigen and the TH1-inducing adjuvants with a solution of the sparingly soluble amino acid or derivative in a strong aqueous acid while neutralizing the mixture of solutions, thereby co-precipitating the sparingly soluble amino acid, antigen and adjuvants. This method may firther comprise adding a pharmaceutically acceptable carrier, diluent or excipient.
The composition of the invention is an antigen-containing formulation comprised of an antigen, a TH-inducing adjuvants, and a sparingly soluble amino acid or a derivative thereof. The antigen-containing formulation is particularly useful as a vaccine; however, the formulation is useful in other contexts as well, e.g., in treating, preventing or reducing susceptibility to certain diseases and disorders, including, but not limited to, bacterial infections, viral infections, and cancer. The individual components of the formulation will now be described by way of non-limiting example.
(A) The Antifen:
Originally the term xe2x80x9cantigenxe2x80x9d was used for any molecule that induced B cells to produce a specific antibody. Now, however, the term may be used to indicate any molecule that can be specifically recognized by the adaptive elements of the immune response, i.e., by B cells or T cells, or both. Thus, an xe2x80x9cantigenxe2x80x9d is a molecule that reacts with preformed antibody at specific receptors on T and B cells. However, we exclude what are traditionally known as xe2x80x9callergens,xe2x80x9d i.e., an agent, e.g., pollen dust, that causes IgE-mediated hypersensitivity.
An allergy is a response to an environmental antigen (allergen) due to pre-existing IgE antibody attached to mast cells. An immediate hypersensitivity reaction is produced by mast cell products (histamine, etc.) causing asthma, hay fever, serum sickness, systematic anaphylaxis or contact dermatitis. There are four types of such hypersensitivity reaction (Types I, II, III and IV). The first three are antibody-mediated; the fourth is mediated mainly by T cells and macrophages. The present invention does not relate to the use of such xe2x80x9cenvironmental antigens.xe2x80x9d
Thus, preferably the xe2x80x9cantigenxe2x80x9d used in the present invention does not include an xe2x80x9callergenxe2x80x9d derived from any allergy causing substance, such as pollen (e.g., ragweed or birch pollen), food, insect venom, mold, animal fur or house dust mite (D. farinae or D. pteronyssinus).
The present invention can therefore be seen as relating to antigens that often involve a cellular, IgG2a or IgG2b mediated response, rather than an IgE or IgG1 mediated response.
The antigen used in the present invention is preferably an immunogen, i.e., an antigen that activates immune cells to generate an immune response against itself.
In a preferred embodiment, the present invention relates to a formulation for use as a vaccine and the antigen is one that is useful in such a vaccine.
The antigen used in the present invention can be any appropriate antigen, which is or becomes available.
The type of antigen used in a vaccine depends on many factors. In general, the more antigens of the microbe retained in the vaccine the better, and living organisms tend to be more effective than killed ones. Exceptions to this rule are diseases where a toxin is responsible for any pathogenic effect. In this case the vaccine can be based on the toxin or toxoid alone.
The antigen used in the present invention may be derived from any living organisms; intact or non-living organisms; subcellular fragments; toxoids; recombinant DNA-based antigens or anti-idiotypes or synthetic antigens. The antigen may be derived from natural or attenuated organisms, which may be viral or bacterial. The type of antigen may be a capsular polysaccharide, surface or internal antigen. If recombinant DNA-based, the antigen may be obtained from a cloned and expressed gene or naked DNA.
The antigen may be modified by reaction, for example, with a cross-linking agent, such as a dialdehyde, more particularly glutaraldehyde.
For example, micro-organisms against which vaccines are available or are sought include Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Proteus, Yersinia, Vibrio, Aeromonas, Pasteurella, Pseudomonas, Acinetobacter, Moraxella, Flavobacterium, Bordetella, Actinobacillus, Neisseria, Brucella, Haemophilus and Escherichia coli. 
Preferred vaccines include, but are not limited to: vaccinia (for smallpox); vole bacillus (for tuberculosis); polio; measles, mumps; rubella; yellow fever; varicella-zoster;
BCG (Bacillus Calmette-Gruxc3xa9rin, an antituberculosis vaccine); rabies; influenza; hepatitis A; typhus; pertussis; typhoid; cholera; plague; pneumococcus; meningococcus; Haemophilus influensae; hepatitis B; hepatitis C; tetanus and diphtheria. Toxin-based vaccines include Chlostridium tetani, Corynebacterium diphtheriae, Vibrio cholerae and Clostridium perfringens. 
Other major diseases for which the vaccines of the invention may be useful include, but are not limited to: HIV, herpes, viruses, adenoviruses, rhinoviruses, staphylococci, group A streptococci, Mycobacterium leprae, Treponema pallidum, Chlamydia, Candida, Pneumocystis, malaria, trypanosomiasis; Chagas"" disease; schistosomiasis and onchoceriasis.
The presence of tumor antigens also has been demonstrated, and, as a result, the concept of vaccinating against cancer has arisen. Also, in principle, conception and implantation can be interrupted by inducing immunity against a wide range of pregnancy and other reproductive hormones.
(B) The TH1-Inducing Adjuvants:
By xe2x80x9cTH1-inducing adjuvantsxe2x80x9d is meant an adjuvants that enhances the TH1 response to an antigen.
The effectiveness of an adjuvants as a TH1-inducing adjuvants may be evaluated by determining the profile of antibodies directed against an antigen resulting from administration of this antigen in vaccines that are also comprised of the various adjuvants.
Preferably the adjuvants is a modified lipopolysaccharide. As described in U.S. Pat. No. 4,912,094, enterobacterial lipopolysaccharide (LPS) is a powerfuil immunostimulant. However, it can also elicit harmfuil and sometimes fatal responses. It is now known that the endotoxic activities associated with LPS result from its lipid A component. Accordingly, the present invention more preferably uses a detoxified derivative of lipid A. Ribi produced a derivative of lipid A originally known as refmed detoxified endotoxin (RDE) but which has become known as monophosphoryl lipid A (MPL). As described in U.S. Pat. No, 4,912,094, MPL is produced by refluxing LPS or lipid A obtained from heptoseless mutants of gram negative bacteria (e.g., Salmonella sp.) in mineral acid solutions of moderate strength (e.g., 0.1N HCl) for a period of around 30 minutes. This treatment results in loss of the phosphate moiety at position 1 of the reducing-end glucosamine. In addition, the core carbohydrate is removed from the 6xe2x80x2 position of the non-reducing glucosamine during this treatment.
Preferably, however, a modified LPS or lipid A is used in which the detoxified lipid A retains the core moiety attached to the 6xe2x80x2 position of non-reducing glucosamine. Such derivatives of LPS and lipid A are also described in U.S. Pat. No. 4,912,094. In more detail, U.S. Pat. No. 4,912,094 discloses a modified lipopolysaccharide that is obtained by selectively removing only the P-hydroxymyristic acyl residue of lipopolysaccharide that is ester-linked to the reducing-end glucosamine at position 3xe2x80x2 of said lipopolysaccharide, which comprises subjecting said lipopolysaccharide to alkaline hydrolysis. Such de-O-acylated monophosphoryl lipid A, diphosphoryl lipid A (DPL) and LPS may be used as the TH1-inducing adjuvants in the present invention. Thus in a preferred embodiment, the present invention uses MPL, DPL or LPS in which the position 3xe2x80x2 of the reducing end glucosamine is de-O-acylated. These compounds are known as 3-DMPL, 3-DDPL and 3-DLPS respectively.
In U.S. Pat. No. 4,987,237 derivatives of MPL having the formula 
are described, wherein R1 and R2 are H or lower alkyl, and R3 is straight or branched chain hydrocarbon composed of C, H and optionally O, N and S, which if more than one atom may be the same or different, wherein the total number of carbon atoms does not exceed 60, and the circle represents an MPL nucleus.
Alternatively, the MPL derivative has the formula 
wherein the segment of the derivative represented by 
contains 2-60 carbon atoms and wherein R3 is straight or branched chain hydrocarbon composed of C, H and optionally O, N and S, which if more than one atom may be the same or different, and x is a minimum of 1 and can be any whole number such that the total number of C atoms in all x segments does not exceed 60, and wherein the chemical structure of each R3 may be the same or different in each such segment and wherein the circle represents an MPL nucleus.
All such derivatives of LPS or lipid A which are or become available may be used as the TH1-inducing adjuvants in the present invention.
The TH1-inducing adjuvants can be mixed with the other components of the composition prior to administration. Alternatively, it can be formulated together with the other components during manufacture of the product. Alternatively, it can be administered at a different site or time than the other components. Administration can be by a number of routes.
(C) The Paringly Soluble Amino Acid:
The amino acid of the present formulation must be sparingly soluble in aqueous solution such that the adjuvants and antigen are allowed to produce an immune response.
Most of the amino acids are only sparingly soluble in water, as a consequence of the strong intermolecular forces acting in the crystal lattice. Exceptions are glycine, proline, lysine, threonine, cysteine and arginine, which are all quite soluble in water, and do not form part of the invention. The water solubility of amino acids is given in the following Table:
Preferably the water solubility of the amino acid used in the invention is about 1.1 or less g/100 ml H2O at 25xc2x0 C. Particularly preferred are tyrosine or tryptophan; the more insoluble tyrosine being preferred. Derivatives of these amino acids such as benzyl-O-octadecanoyl-L-tyrosine are also included within the scope of the present invention.
Typically, the antigen is dispersed within and/or adsorbed onto the amino acid, e.g., by co-precipitation or mixing, respectively.
The composition of the present invention may be prepared by mixing an aqueous solution of the antigen with a solution of the amino acid in a strong aqueous acid, neutralizing the mixture of solution, thereby co-precipitating the amino acid and antigen, mixing the product with the TH1-inducing adjuvants, and optionally adding a physiologically acceptable diluent, excipient or carrier, before or after the aforementioned mixture. Alternatively, the TH1-inducing adjuvants may be co-precipitated with the antigen. As well as being mixed or co-precipitated with the other components of the composition prior to administration, the TH1-inducing adjuvants can be administered at a different site and/or time to the other components.
Typically an aqueous solution of the antigen, preferably at pH 7xc2x11, obtainable from the solvation of a solid, is mixed with a solution of the amino acid in a strong aqueous acid. The strong acid is usually an inorganic acid, preferably hydrochloric acid. The solution of antigen used in this step typically contains between 0.1 xcexcg/ml and 1000 xcexcg/ml antigen protein, for example about 400 xcexcg/ml. The ratio of antigen:amino acid in the mixture is typically in the range of approximately 1:4xc3x97105 to 1:1xc3x97102 W/W.
The resulting mixture of solutions of antigen and amino acid is neutralized. By neutralization is meant an adjustment of pH to a value within the range 4.0 to 7.5. It is desirable that, at no time, or at least no prolonged time, during the neutralization does the pH of the solution rise appreciably above 7.5. This condition can be met by vigorous stirring of the solution and by the use of only the required amount of base, if desired. Various buffering agents can usefully be added to the solutions of antigen to assist in pH control during mixing and neutralizing stages.
A particularly useful method of carrying out the neutralization is for separate streams of the solution of amino acid and neutralizing base to be run into the solution of antigen. The rates of flow of the added solutions are controlled by pH-state, that is, by equipment that regulates the flow of one or both of the solutions so that the pH of the reaction mixture remains substantially constant at a predetermined level. Optimum results are generally obtained by maintaining the pH within the range of approximately 6.5 to 7.5, although the precise pH may vary according to the nature of the antigen.
The result of neutralization is the immediate precipitation of the amino acid, within and/or upon which the solution of antigen is occluded and/or adsorbed. After precipitation, the mixture is either washed immediately or allowed to stand for a period of from a few hours to a day or two prior to washing.
The resulting precipitate may be removed from the solution by centrifugation or filtration and washed, e.g., with phenol-saline, before resuspending, if required, in a physiologically acceptable carrier, excipient or diluent.
MPL (or other TH1-inducing adjuvants) that has been dissolved by the method described in Preparation 3 below or by sonification can be diluted by various means prior to its addition to amino acid adsorbates of antigens. The preparation of MPL is initially made at a concentration of typically between 0.5 mg per ml and 4 mg per ml, for example 1 mg per ml. It can then be diluted to a concentration of between 500 pg/ml and 20 jg/ml, preferably 100 xcexcg/ml. This dilution can be made in pure water, or in an aqueous glycerol solution containing between 1% and 4%, preferably 2%, glycerol. Such dilutions can then be added to a suspension of the amino acid adsorbate prepared as described above. For convenience, the concentration of the MPL solution and the amino acid adsorbate suspension respectively may be selected such that approximately equal volumes of each of admixed to obtain the final product for injection. A typical final product contains about 100 xcexcg/ml of antigen and about 250 xcexcg/ml of MPL.
Thus, although the formulation of the invention may be administered directly, preferably the formulation is combined with a pharmaceutically acceptable carrier, excipient or diluent to produce a pharmaceutical composition, which may be for human or veterinary use. Suitable physiologically acceptable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline, phenol-saline and sterile water. The compositions may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
The routes of administration and dosages described herein are intended only as a guide since a skilled practitioner will readily be able to determine the optimum route of administration and dosage for any particular patient and condition.
Vaccines may be prepared from the formulation of the present invention. The preparation of vaccines that contain an antigen as active ingredient is known to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the formulation encapsulated in liposomes. As indicated above, the formulation may be mixed with carriers, diluents and excipients that are pharmaceutically acceptable and compatible with the formulation. Such excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and/or further adjuvants that enhance the effectiveness of the vaccine.
The proportion of antigen and adjuvants can be varied over a broad range so long as both are present in effective amounts. Conveniently, the vaccines are formulated to contain a final concentration of antigen in the range of 0.2 xcexcg/ml to 200 xcexcg/ml, preferably 5 xcexcg/ml to 50 xcexcg/ml, most preferably about 15 xcexcg/ml.
After formulation, the vaccine may be incorporated into a sterile container that is then sealed and stored at low temperature, for example 40xc2x0 C., or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the vaccine composition is lyophilized, the lyophilized material may be reconstituted prior to administration, e.g., as a suspension. Reconstitution is preferably effected in buffer.
Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, copolymers of methacrylic acid and methyl methacrylate available under the trademark Eudragit S, and Eudragit L, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
The antigens used in the invention may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5 xcexcg to 250 xcexcg of antigen per dose, depends on the subject to be treated, capacity of the subject""s immune system to synthesize antibodies, and the degree of protection desired. A preferable range is from about 20 xcexcg to about 40 xcexcg per dose.
A suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 20 xcexcg of immunogen in admixture with 0.5% adjuvants.
Precise amounts of active ingredient required to be administered will depend on the judgment of the practitioner and may vary with each subject.
The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgement of the practitioner.
In addition, the vaccine containing the antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins.
Compositions according to the invention may be used directly as immunogens, without the use of further adjuvants to generate antisera and monoclonal antibodies. The invention thus provides a method for inducing antigen specific immunoglobulin production comprising the steps of:
a) immunizing an animal with a composition according to the present invention; and
b) recovering immunoglobulin specific for a region of the antigen of the composition from the serum of the animal.
The animals used for antibody production may be any animals normally employed for the purpose, particularly mammals. Especially indicated are mice, rats, guinea pigs and rabbits.
Immunization is carried out according to established techniques (See xe2x80x9cAntibodies, A Laboratory Manualxe2x80x9d by E. Harlow and D. Lane (1988) Cold Spring Harbor, U.S.A.). The purified composition (about 1 mg) was injected into a rabbit. A booster injection of 0.5 mg of the composition was made 4 weeks after the initial injection. Antibodies are isolated from rabbit serum and tested for reactivity. Antibodies capable of selective binding to the chosen antigen are obtained by this method.
More particularly, the formulation of the present invention comprising the antigen can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
Monoclonal antibodies directed against antigens used in the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against antigens can be screened for various properties; i.e., for isotype and epitope affinity.
An alternative technique involves screening phage display libraries where, for example, the phage express scFv fragments on the surface of their coat with a large variety of complementary determining regions (CDRs). This technique is well known in the art.
Antibodies, both monoclonal and polyclonal, which are directed against antigens are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins that carry an xe2x80x9cinternal imagexe2x80x9d of the antigen of the infectious agent against which protection is desired.
Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful for treatment, as well as for an elucidation of the immunogenic regions of antigens.
For the purposes of this invention, the term xe2x80x9cantibody,xe2x80x9d unless specified to the contrary, includes fragments of whole antibodies that retain their binding activity for a target antigen. Such fragments include Fv, F(abxe2x80x2) and F(abxe2x80x2)2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanized antibodies, for example as described in EP-A-239400.
It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the description above as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.